Nhe1 Is Essential for Potassium but Not Calcium Facilitation of Cell Motility and the Monovalent Cation Requirement for Chemotactic Orientation in Dictyostelium discoideum

Abstract

In Dictyostelium discoideum, extracellular K ؉ or Ca 2؉ at a concentration of 40 or 20 mM, respectively, facilitates motility in the absence or presence of a spatial gradient of chemoattractant. Facilitation results in maximum velocity, cellular elongation, persistent translocation, suppression of lateral pseudopod formation, and myosin II localization in the posterior cortex. A lower threshold concentration of 15 mM K ؉ or Na or 5 mM Ca 2؉ is required for chemotactic orientation. Although the common buffer solutions used by D. discoideum researchers to study chemotaxis contain sufficient concentrations of cations for chemotactic orientation, the majority contain insufficient levels to facilitate motility. Here it has been demonstrated that Nhe1, a plasma membrane protein, is required for K ؉ but not Ca 2؉ facilitation of cell motility and for the lower K ؉ but not Ca 2؉ requirement for chemotactic orientation.Extracellular Ca 2ϩ and K ϩ , ubiquitous in the soluble environment of cells, both free-living and in multicellular organisms (5,15,21,36,56,79,80,84,91), have long been known to affect the motility of cells ranging in complexity from bacterial to human (7,8,19,25,31,60,62,65,98). In Dictyostelium discoideum, which serves as a powerful model for studying animal cell motility (4,6,20,34,41,42,43,45,50,82,87,86,92,98), either extracellular K ϩ or Ca 2ϩ at an optimum concentration of 40 mM or 20 mM, respectively, enhances most aspects of basic cell motility in the absence of chemoattractant, including cell elongation, uropod formation, the suppression of lateral pseudopod formation, velocity, directional persistence, and localization of myosin II in the posterior cell cortex (51, 78). These facilitating concentrations of K ϩ or Ca 2ϩ cannot be replaced with other monovalent or divalent cations (51) and are within the range of the soluble concentrations of the two cations found in soil (1,5,32,44,49,56) and manure (9, 66, 83), two common niches in which soil amoebae thrive (12,27,35,67,71). These concentrations of K ϩ and Ca 2ϩ are close to those found to be optimum for enhancing the motility of a variety of other animal cells. For instance, 9 mM Ca 2ϩ has been found to be optimum for flagellated sea urchin sperm motility (99), and Ca 2ϩ ranging in concentration from 1 to 10 mM has been found optimum for vertebrate cell motility (28,58,63). Concentrations of K ϩ greater than 100 mM have been found to be optimum for the polarization and motility of human polymorphonuclear leukocytes (52, 69).The surface molecules and mechanisms regulating K ϩ and Ca 2ϩ facilitation in D. discoideum have not been elucidated. However, Patel and Barber (64) reported a defective behavioral phenotype for cells of the nhe1 null mutant in a facilitating concentration of K ϩ that was strikingly similar to the behavior of wild-type cells in a nonfacilitating concentration of K ϩ (51). Nhe1 is a plasma membrane protein related to cation/H ϩ exchangers (64). We therefore tested the possibility that Nhe1 mediated K ϩ facilitation. First, t...

The IplA Ca++ Channel OfDictyostelium discoideumIs Necessary For Ca++, But Not cAMP Chemotaxis, And Plays A Fundamental Role In Natural Aggregation

“…These results are consistent with previous observations, especially on lateral pseudopodium formation and turning, indicating that a spatial gradient of cAMP enhances the effect of Ca 2+ (Lusche et al, 2009). As previously reported (Lusche et al, 2009;Lusche et al, 2011;Scherer et al, 2010), when Ca 2+ was not added (0 mM Ca 2+ ) to a spatial gradient of cAMP generated in TB, Ax2 cells still underwent chemotaxis, but with a reduced chemotactic index (Lusche et al, 2009;Lusche et al, 2011;Scherer et al, 2010). Although relatively mobile, iplA -cells exhibited negligible chemotactic orientation (CI5+0.0160.30; percentage positive chemotaxis570%) in a spatial gradient of cAMP at 0 mM Ca 2+ , but exhibited maximum chemotactic orientation (CI5+0.7560.14), similar to that of Ax2 cells, when the concentration of Ca 2+ was raised to 5 mM ( Fig.…”

“…3A,D Table S1). And as previously reported (Lusche et al, 2009;Lusche et al, 2011), increasing the Ca 2+ concentration in TB from 0 to 5 mM in the (Furuichi et al, 1989;Joseph, 1996;Foskett et al, 2007;Foskett, 2010;Mignery and Sudhof, 1990 absence of cAMP resulted in an increase in the directional persistence of translocating cells (Fig. 3A,E concentration in TB was increased to 10 mM ( Fig.…”

“…As we previously reported for parental Ax2 cells (Lusche et al, 2009;Lusche et al, 2011), increasing the concentration of Ca 2+ in Tricine buffer (TB) from 5 to 10 mM, in the absence of cAMP, resulted in dramatic increases in the three measured cell motility parameters: instantaneous velocity (Fig. 3A,B), the proportion of cells with instantaneous velocities of $9 mm/minute (Fig.…”

“…The N-terminal fusion of GFP to iplA + in pDM351-iplA was confirmed by sequencing. The plasmid pDM351-iplA + was transformed into iplA -cells as described previously Wessels et al, 2007) Aggregation-competent cells were washed in Tricine buffer (TB) and incubated for 1 hour in TB containing 10 mM Ca 2+ (Lusche et al, 2009;Scherer et al, 2010;Lusche et al, 2011). Cells were then washed in TB + 10 mM Ca 2+ and 6610 6 cells incubated in 5 mM NaN 3 in TB.…”

“…For that reason, they must be present at optimum concentrations in the natural environment . For the soil amoeba Dictyostelium discoideum to achieve maximum velocity either the extracellular concentration of Ca 2+ must reach 10 mM or the concentration of K + reach 40 mM (Lusche et al, 2009;Lusche et al, 2011;Scherer et al, 2010). Moreover, for D. discoideum amoebae to orient in a spatial gradient of the chemoattractant cAMP, either Ca 2+ must reach 5 mM, or K + or Na + reach 15 mM (Lusche et al, 2009;Lusche et al, 2011).…”